The genetic code is degenerate. With the exception of two amino acids (methionine and tryptophan), all other amino acid residues are each encoded by multiple, so-called synonymous codons. Synonymous codons are however not present at equal frequencies in individual mRNAs as well as entire genomes. This pattern of non- uniform codon use is known as codon usage bias. Codon usage bias varies between organisms and represents a unique feature of an organism. Organism-specific codon choice is related to organism-specific differences in populations of cognate tRNAs. In both unicellular and multicellular organisms there exists a strong positive correlation between codon usage and cellular tRNA content, meaning that codon bias would likely have a direct impact on translation elongation rates. Indeed, frequently used/optimal codons were, as a rule, are found to be translated more rapidly than infrequently used ones due to the higher availability (during translation) of corresponding frequent cognate tRNAs. A technological implication of codon bias is that substitution of rare synonymous codons with frequently used ones (codon optimization), can increase protein synthesis rates and thus protein yield. This platform technology has been widely used in basic research and in biotechnology industry for production of recombinant/therapeutic-proteins. However, numerous recent studies identified an important drawback of this standard approach for codon optimization. Although, synonymous changes were presumed to be silent, recent data have shown that synonymous codon substitutions may influence many aspects of mRNA and protein biogenesis. Importantly, it was demonstrated that synonymous codon substitutions may affect protein folding and post-translational modifications and thus may have functional consequences. Furthermore, synonymous codon changes were found to be associated with over 50 diseases unequivocally demonstrating the importance of codon usage for gene/protein function. However, currently, there is yet a limited understanding of why some synonymous mutations have functional and clinical consequences while others do not. The proposed studies are aimed at elucidating the effects of synonymous codon substitutions on protein function, using blood coagulation factor IX (FIX), coded by F9 gene as a model system. Genetic defects in F9 are responsible for hemophilia B; while several disease-associated synonymous mutations have been identified in this gene. Moreover, FIX is a drug-product amenable to codon-optimization and codon-optimized versions of F9 are used in gene therapy trials. Our goal is to use in vitro and ex vivo approaches to assess and understand the effects of codon optimization on FIX folding and function, and define regions in F9 mRNA in which synonymous mutations would be deleterious. Also, immunogenicity is another key concern in the development of any therapeutic protein; however, the potential influence of codon- optimization on eliciting immune responses has not been studied. By assessing the peptides presented on the surface of antigen presenting cells, through a MHC-associated peptide proteomics (MAPPS) assay, we generated preliminary data indicating that wild-type and codon-optimized FIX variants are processed and presented differently. We will further examine the propensity of the identified FIX-peptides to induce an immune response by investigating T cells responses. Data from functional analysis and immunogenicity studies will be combined to define the best codon optimization strategies that provide the highest yields of fully functional protein with unaltered immunogenicity, thus allowing the creation of safer and more effective FIX therapeutics. We believe this approach will be amenable to the design of any protein therapeutic.